The results of a careful electon microscope study of NK and ADCC lysis of target cells had suggested that the contents of cytoplasmic granules were secreted in the vicinity of target cells during the cytotoxic process. These findings are strongly supported by the recent experiments described here in which we have demonstrated the presence of a very potent lytic agent in cytoplasmic granules purified from rat NK tumors. The activity of this lytic agent is controlled by calcium which is absolutely required (less than 10 to the -4M) for its expression. Magnesium is totally inactive. The lytic activity is most easily demonstrated in red cell targets (human or sheep), where less than 1 Mug/ml granule protein suffices to lyse 100% of the target cells in less than 15 minutes. This is less than one equivalent Nk cell per target cell. When red cell ghosts are treated under these conditions they can be seen to remain intact, and fluorescence microscopy reveals that very large molecules (less than 500,000 MW) remain inside while small molecules are released. This is evidence for aqueous pores in the ghost membranes, similar to the membrane damage inflicted by intact effector cells. Granule preparations from noncytotoxic cells do not show cytolytic activity.